The amount of cholesterol present in the blood is known to be related to the risk of coronary artery disease. Cholesterol circulates in the blood predominantly in protein-bound form The proteins which transport cholesterol are the lipoproteins, which are subdivided into classes based on their density. High-density lipoproteins (HDL), which typically account for about 20-30% of serum cholesterol, are involved in the catabolism of triglyceride-rich lipoproteins and in the removal of cholesterol from peripheral tissues and transport to the liver. The very-low density lipoproteins (VLDL) are triglyceride-rich lipoproteins which are synthesized in the liver and ultimately converted to low-density lipoproteins (LDL), which transport most of the plasma cholesterol in humans. Chylomicrons are a type of very low density lipoproteins that are synthesized in the intestinal mucosa and transport exogenous (dietary) cholesterol and triglycerides from the small intestine to muscle and adipose tissues. VLDL's typically account for about 5% of total serum cholesterol, while LDL's typically account for about 60-75%. However, diets high in saturated fat and cholesterol can cause an increase in the amount of LDL cholesterol in the blood.
A relationship between serum levels of different lipoproteins and risk of coronary disease has been established. In particular, if the proportion of serum cholesterol associated with LDL is high and/or the proportion associate with HDL is low, the risk of coronary disease is increased.
In view of the importance of relative serum cholesterol levels in risk assessment and management of atherogenic disease, considerable effort has been spent screening large populations of both normal and high-risk individuals for serum levels of HDL, LDL, as well as total cholesterol and triglycerides. The effectiveness of treatments of high-risk individuals has been monitored by regular testing of serum levels of cholesterol in the various lipoprotein compartments.
LDL-associated lipoprotein is often measured indirectly by separately determining total serum cholesterol, triglycerides, and HDL associated cholesterol. Various methods have been proposed for direct quantification of LDL. The different lipoproteins can be separated by ultracentrifugation, which is a generally accurate method for determining LDL, but impractical for clinical use. A series of patents by Ziegenhorn et al. (U.S. Pat. No. 4,746,605 (1988), U.S. Pat. No. 5,407,836 (1995), and U.S. Pat. No. 5,532,172 (1996)) describe a method in which HDL is removed from a serum sample by addition of an anti-HDL antibody, and triglyceride-rich very low density lipoproteins (VLDL) are removed by precipitation with an anionic polymer, e.g. dextran sulfate, leaving LDL in solution for independent quantification. However, this method requires manipulation of several solutions and typically employs centrifugation to remove the antibody-bound or precipitated lipoproteins.
Reports by Sugiuchi et al. (1998) and Nauck et al. (2000) describe a homogenous solution assay in which an anionic polymer, e.g. cyclodextrin sulfate, is used to reduce reactivity of VLDL and chylomicron cholesterol without precipitation, while a nonionic surfactant such as PEO/PPO is used to solubilize, and thus increase the reactivity of, cholesterol in LDL particles. The Direct LDL™ assay developed by Genzyme (see e.g. NcNamara et al., 1995; Cole, 1996) employs antibodies directed to VLDLs and HDLs to bind these lipoproteins, followed by centrifuging and filtering to recover the supernatant containing LDL.
These liquid-phase assays have a number of limitations with respect to their use in widespread screening. First, the methods generally require a venous blood sample, requiring a trained technician to draw, fractionate and aliquot the blood sample. The sample must be treated with reagents such as precipitating agent, binding agent, surfactant, and, in most cases, further processed to remove precipitated material. Although some of these procedures can be automated, analytical machines designed for this purpose are expensive and not widely available outside of large hospitals.
Law et al. (U.S. Pat. No. 5,411,870) describe a device for use in direct determination of LDL which does not require wet chemistry manipulation of the sample. However, the device requires a dissolvable layer which separates “LDL redissolution agents” from a zone containing soluble HDL and VLDL and precipitated LDL. The device relies on dissolution of the separating layer, which allows LDL in the first zone to be dissolved, after a time sufficient to allow HDL and VLDL in the first zone to be degraded by enzymes, and the hydrogen peroxide thereby generated to be consumed by catalase. It can be seen that timing of this dissolution would be critical to obtaining accurate quantification of LDL cholesterol.
It is therefore the object of the present invention to provide an LDL assay device and method which overcome the above-noted prior art disadvantages.